Korean Red Ginseng Extract Inhibits IL-8 Expression via Nrf2 Activation in Helicobacter pylori-Infected Gastric Epithelial Cells.

Helicobacter pylori (H. pylori) causes gastric diseases by increasing reactive oxygen species (ROS) and interleukin (IL)-8 expression in gastric epithelial cells. ROS and inflammatory responses are regulated by the activation of nuclear factor erythroid-2-related factor 2 (Nrf2) and the expression of Nrf2 target genes, superoxide dismutase (SOD) and heme oxygenase-1 (HO-1). We previously demonstrated that Korean red ginseng extract (RGE) decreases H. pylori-induced increases in ROS and monocyte chemoattractant protein 1 in gastric epithelial cells. We determined whether RGE suppresses the expression of IL-8 via Nrf2 activation and the expression of SOD and HO-1 in H. pylori-infected gastric epithelial AGS cells. H. pylori-infected cells were treated with RGE with or without ML385, an Nrf2 inhibitor, or zinc protoporphyrin (ZnPP), a HO-1 inhibitor. Levels of ROS and IL-8 expression; abundance of Keap1, HO-1, and SOD; levels of total, nuclear, and phosphorylated Nrf2; indices of mitochondrial dysfunction (reduction in mitochondrial membrane potential and ATP level); and SOD activity were determined. As a result, RGE disturbed Nrf2-Keap1 interactions and increased nuclear Nrf2 levels in uninfected cells. H. pylori infection decreased the protein levels of SOD-1 and HO-1, as well as SOD activity, which was reversed by RGE treatment. RGE reduced H. pylori-induced increases in ROS and IL-8 levels as well as mitochondrial dysfunction. ML385 or ZnPP reversed the inhibitory effect of RGE on the alterations caused by H. pylori. In conclusion, RGE suppressed IL-8 expression and mitochondrial dysfunction via Nrf2 activation, induction of SOD-1 and HO-1, and reduction of ROS in H. pylori-infected cells.

Helicobacter pylori and Epstein-Barr Virus Coinfection Stimulates Aggressiveness in Gastric Cancer through the Regulation of Gankyrin.

Persistent coinfection with Helicobacter pylori and Epstein-Barr virus (EBV) promotes aggressive gastric carcinoma (GC). The molecular mechanisms underlying the aggressiveness in H. pylori and EBV-mediated GC are not well characterized. We investigated the molecular mechanism involved in H. pylori- and EBV-driven proliferation of gastric epithelial cells. Results showed that the coinfection is significantly more advantageous to the pathogens as coinfection creates a microenvironment favorable to higher pathogen-associated gene expression. The EBV latent genes ebna1 and ebna3c are highly expressed in the coinfection compared to lone EBV infection at 12 and 24 h. The H. pylori-associated genes 16S rRNA, cagA, and babA were also highly expressed during coinfection compared to H. pylori alone. In addition, upregulation of gankyrin, which is a small oncoprotein, modulates various cell signaling pathways, leading to oncogenesis. Notably, the knockdown of gankyrin decreased the cancer properties of gastric epithelial cells. Gankyrin showed a similar expression pattern as that of ebna3c at both transcript and protein levels, suggesting a possible correlation. Further, EBV and H. pylori created a microenvironment that induced cell transformation and oncogenesis through dysregulation of the cell cycle regulatory (ccnd1, dapk3, pcna, and akt), GC marker (abl1, tff-2, and cdx2), cell migration (mmp3 and mmp7), DNA response (pRB, pten, and p53), and antiapoptotic (bcl2) genes in infected gastric epithelial cells through gankyrin. Our study provides a new insight into the interplay of two oncogenic agents (H. pylori and EBV) that leads to an enhanced carcinogenic activity in gastric epithelial cells through overexpression of gankyrin. IMPORTANCE In the present study, we evaluated the synergistic effects of EBV and H. pylori infection on gastric epithelial cells in various coinfection models. These coinfection models were among the first to depict the exposures of gastric epithelial cells to EBV followed by H. pylori; however, coinfection models exist that narrated the scenario upon exposure to H. pylori followed by that to EBV. We determined that a coinfection by EBV and H. pylori enhanced the expression of oncogenic protein gankyrin. The interplay between EBV and H. pylori promoted the oncogenic properties of AGS cells like elevated focus formation, cell migration, and cell proliferation through gankyrin. EBV and H. pylori mediated an enhanced expression of gankyrin, which further dysregulated cancer-associated genes such as cell migratory, tumor suppressor, DNA damage response, and proapoptotic genes.

Virus-host interactions in carcinogenesis of Epstein-Barr virus-associated gastric carcinoma: Potential roles of lost ARID1A expression in its early stage.

Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is a distinct molecular subtype of gastric cancer characterized by viral infection and cellular abnormalities, including loss of AT-rich interaction domain 1A (ARID1A) expression (lost ARID1A). To evaluate the significance of lost ARID1A in the development of EBVaGC, we performed in situ hybridization of EBV-encoded RNA (EBER) and immunohistochemistry of ARID1A in the non-neoplastic gastric mucosa and intramucosal cancer tissue of EBVaGC with in vitro infection analysis of ARID1A-knockdown and -knockout gastric cells. Screening of EBER by in situ hybridization revealed a frequency of approximately 0.2% EBER-positive epithelial cells in non-neoplastic gastric mucosa tissue samples. Six small foci of EBV-infected epithelial cells showed two types of histology: degenerated (n = 3) and metaplastic (n = 3) epithelial cells. ARID1A was lost in the former type. In intramucosal EBVaGC, there were ARID1A-lost (n = 5) and -preserved tumors (n = 7), suggesting that ARID1A-lost carcinomas are derived from ARID1A-lost precursor cells in the non-neoplastic mucosa. Lost ARID1A was also observed in non-neoplastic mucosa adjacent to an ARID1A-lost EBVaGC. In vitro experiments using siRNA knockdown and the CRISPR/Cas9-knockout system demonstrated that transient reduction or permanent loss of ARID1A expression markedly increased the efficiency of EBV infection to stomach epithelial cells. Taken together, lost ARID1A plays a role in initiating EBV-driven carcinogenesis in stomach epithelial cells, which develop to a distinct subtype of EBVaGC within the proper mucosal layer. Lost ARID1A is one of the constituents of virus-host interactions in the carcinogenesis of EBVaGC.

Circulating integrin α4 ß7+ CD4 T cells are enriched for proliferative transcriptional programs in HIV infection.

HIV preferentially infects α4 ß7+ CD4 T cells, forming latent reservoirs that contribute to HIV persistence during antiretroviral therapy. However, the properties of α4 ß7+ CD4 T cells in blood and mucosal compartments remain understudied. Employing two distinct models of HIV infection, HIV-infected humans and simian-human immunodeficiency virus (SHIV)-infected rhesus macaques, we show that α4 ß7+ CD4 T cells in blood are enriched for genes regulating cell cycle progression and cellular metabolism. Unlike their circulating counterparts, rectal α4 ß7+ CD4 T cells exhibited a core tissue-residency gene expression program. These features were conserved across primate species, indicating that the environment influences memory T-cell transcriptional networks. Our findings provide an important molecular foundation for understanding the role of α4 ß7 in HIV infection.

Gut Susceptibility to Viral Invasion: Contributing Roles of Diet, Microbiota and Enteric Nervous System to Mucosal Barrier Preservation.

The gastrointestinal lumen is a rich source of eukaryotic and prokaryotic viruses which, together with bacteria, fungi and other microorganisms comprise the gut microbiota. Pathogenic viruses inhabiting this niche have the potential to induce local as well as systemic complications; among them, the viral ability to disrupt the mucosal barrier is one mechanism associated with the promotion of diarrhea and tissue invasion. This review gathers recent evidence showing the contributing effects of diet, gut microbiota and the enteric nervous system to either support or impair the mucosal barrier in the context of viral attack.

LGR5 expression is associated with prognosis in poorly differentiated gastric adenocarcinoma.

BACKGROUND: Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is an important cancer stem cell marker in gastric cancer. However, no detailed studies are available on LGR5 expression in poorly differentiated gastric adenocarcinoma (PD-AC). Therefore, we investigated the relationship between LGR5 expression and clinicopathological data in PD-AC. METHODS: LGR5 mRNA expression levels were quantified in 41 PD-AC specimens using a highly sensitive RNAscope in situ hybridization technique. Epstein-Barr virus (EBV) infection was also detected by EBV in situ hybridization. RESULTS: LGR5 expression levels were measured in 38 of 41 PD-AC cases, and 17 cases were identified as LGR5 high. The frequency of EBV positivity tended to be higher in the LGR5-low group than in the LGR5-high group (P = 0.0764). Furthermore, the frequency of vascular invasion tended to be higher in the LGR5-high group than in the LGR5-low group (P = 0.0764). The overall survival of PD-AC patients in the LGR5-high group was significantly lower than in the LGR5-low group (log-rank test, P = 0.0108). The Cox proportional hazard regression model revealed that the LGR5-low group (HR = 0.29; 95% CI: 0.11-0.74; P = 0.01) showed independently better OS for PD-AC. CONCLUSIONS: Quantifying the levels of LGR5 expression may facilitate defining prognosis in Japanese patients with PD-AC. Further study of LGR5 in this context is warranted.

Targeting of Mammalian Glycans Enhances Phage Predation in the Gastrointestinal Tract.

The human gastrointestinal mucosal surface consists of a eukaryotic epithelium, a prokaryotic microbiota, and a carbohydrate-rich interface that separates them. In the gastrointestinal tract, the interaction of bacteriophages (phages) and their prokaryotic hosts influences the health of the mammalian host, especially colonization with invasive pathobionts. Antibiotics may be used, but they also kill protective commensals. Here, we report a novel phage whose lytic cycle is enhanced in intestinal environments. The tail fiber gene, whose protein product binds human heparan sulfated proteoglycans and localizes the phage to the epithelial cell surface, positions it near its bacterial host, a type of locational targeting mechanism. This finding offers the prospect of developing mucosal targeting phage to selectively remove invasive pathobiont species from mucosal surfaces.IMPORTANCE Invasive pathobionts or microbes capable of causing disease can reside deep within the mucosal epithelium of our gastrointestinal tract. Targeted effective antibacterial therapies are needed to combat these disease-causing organisms, many of which may be multidrug resistant. Here, we isolated a lytic bacteriophage (phage) that can localize to the epithelial surface by binding heparan sulfated glycans, positioning it near its host, Escherichia coli This targeted therapy can be used to selectively remove invasive pathobionts from the gastrointestinal tract, preventing the development of disease.

Gastritis cystica profunda is associated with aberrant p53 and Epstein-Barr virus in gastric cancer: A clinicopathological, immunohistochemical and in situ hybridization study.

Gastritis cystica profunda (GCP) is a lesion characterized by cystic gastric glands within the submucosa. Some studies have reported that GCP is a precancerous lesion. Here, we investigated the association between GCP and gastric cancer. Gastric cancer specimens were taken from 1432 patients undergoing surgery or endoscopic submucosal resection and were classified as GCP or non-GCP. The clinicopathological features, immunohistochemistry and in situ hybridization expression of p53, Ki-67, KCNE2, Epstein-Barr virus (EBV) and programmed death ligand 1 (PD-L1) were compared between the two groups, as well as between GCPs and normal pyloric glands. One hundred and eighty patients (12.6%) had GCPs. In the GCP group, no cancerous lesions were found within the GCPs, but 13% were linked to GCPs and 60.2% were located above or near GCPs. Aberrant p53 expression, EBV-positive cancer cells and PD-L1 scores were significantly higher in the GCP group. The p53 score and Ki-67 labelling index were significantly higher and the KCNE2 score was significantly lower in GCPs than in pyloric glands. Although we suggest GCP is paracancerous, GCP has high proliferation activity and gastric cancer with GCP is associated with aberrant p53 and EBV. GCP is associated with aberrant p53 expression and EBV.

The role of the Epstein-Barr virus-encoded BARF1 gene expressed in human gastric epithelial cells.

BACKGROUND/AIMS: The study aimed to explore the effects of Epstein-Barr virus--encoded BARF1 in human gastric epithelial cells (GES-1). MATERIALS AND METHODS: A eukaryotic expression vector carrying BARF1 gene (pcDNA3.1-BARF1) was constructed. The pcDNA3.1-BARF1 was transfected into GES-1 cells, and they were selected by G418. The GES-1 cells lines that expressed BARF1 (GES-1-BARF1) were obtained. The cycle of GES-1-pcDNA3.1 cells (GES-1 cells transfected with empty vector), GES-1-BARF1 cells (GES-1 cells transfected with BARF1), and TPA-GES-1-BARF1(GES-1-BARF1 cells stimulated by 12-O-tetradecanoylphorbol-13-acetate (TPA) were analyzed by flow cytometry. Colony formation in soft agar and tumorigenicity of the transfected cells in mice with severe combined immunodeficiency (SCID) were also observed. RESULTS: The morphology of GES-1-BARF1 cells were changed from the original shuttle to round, the adhesion between the cells and bottle wall was weakened, and the cells showed overlapping growth. The proliferation rate of GES-1-BARF1 and TPA-GES-1-BARF1 cells were faster than GES-1 and GES-1-pcDNA3.1 cells; the S phase was significantly prolonged for GES-1-BARF1 and TPA-GES-1-BARF1. GES-1-BARF1 and TPA-GES-1-BARF1 cells formed colonies in soft agar, with a cloning rate of 24.2% (58/240) and 40.0% (96/240), respectively; GES-1 and GES-1-pcDNA3.1 cells did not form colonies in soft agar. Tumors were formed in mice with SCID after injecting TPA-GES-1-BARF1 cell groups. Tumor formation did not occur in mice with SCID after injecting GES-1 and GES-1-pcDNA3.1 cell groups, but nodules were formed in the mice with SCID after injecting GES-1-BARF1 cell groups. CONCLUSION: GES-1-BARF1 cells malignant transformation was induced by transfected BARF1 gene and TPA stimulation. This result indicated that tumor formation not only require oncogenes, but also the stimulation of cancer-promoting substance.

Status of kinases in Epstein-Barr virus and Helicobacter pylori Coinfection in gastric Cancer cells.

BACKGROUND: Helicobacter pylori (H. pylori) and Epstein - Barr virus (EBV) plays a significant role in aggressive gastric cancer (GC). The investigation of genes associated with these pathogens and host kinases may be essential to understand the early and dynamic progression of GC. AIM: The study aimed to demonstrate the coinfection of EBV and H. pylori in the AGS cells through morphological changes, expression of the kinase and the probable apoptotic pathways. METHODS: Genomic DNA isolation of H. pylori and its characterization from clinical samples were performed. RT-qPCR of kinases was applied to scrutinize the gene expression of kinases in co-infected GC in a direct and indirect (separated through insert size 0.45 µm) H. pylori infection set up. Morphological changes in co-infected GC were quantified by measuring the tapering ends of gastric epithelial cells. Gene expression profiling of apoptotic genes was assessed through RT-qPCR. RESULTS: An interleukin-2-inducible T-cell kinase (ITK) showed significant upregulation with indirect H. pylori infection. Moreover, Ephrin type-B receptor six precursors (EPHB6) and Tyrosine-protein kinase Fyn (FYN) showed significant upregulation with direct coinfection. The tapering ends in AGS cells were found to be extended after 12 h. A total of 24 kinase genes were selected, out of which EPHB6, ITK, FYN, and TYK2 showed high expression as early as 12 h. These kinases may lead to rapid morphological changes in co-infected gastric cells. Likewise, apoptotic gene expression such as APAF-1 and Bcl2 family genes such as BAD, BID, BIK, BIM, BAX, AND BAK were significantly down-regulated in co-infected AGS cells. CONCLUSION: All the experiments were performed with novel isolates of H. pylori isolated from central India, for the functional assessment of GC. The effect of coinfection with EBV was more profoundly observed on morphological changes in AGS cells at 12 h as quantified by measuring the tapering of ends. This study also identifies the kinase and apoptotic genes modulated in co-infected cells, through direct and indirect approaches. We report that ITK, EPHB6, TYK2, FYN kinase are enhanced, whereas apoptotic genes such as APAF-1, BIK, FASL, BAX are significantly down-regulated in AGS cells coinfected with EBV and H. pylori.

Associations Between Gastric Cancer Risk and Virus Infection Other Than Epstein-Barr Virus: A Systematic Review and Meta-analysis Based on Epidemiological Studies.

INTRODUCTION: Besides Helicobacter pylori and Epstein-Barr virus, other viruses might play potential roles in gastric carcinogenesis. This systematic review and meta-analysis was conducted to compare the prevalence of the viruses between gastric cancer (GC) and any controls. METHODS: Comprehensive literature was searched up to January 25, 2019, and search was updated on April 6, 2020. The studies that compared the prevalence of viruses other than Epstein-Barr virus between GC and healthy or nonmalignant controls were eligible. Stata 12.0 software was used for heterogeneity tests and meta-analyses. Meanwhile, subgroup analysis, sensitivity analysis, and publication bias evaluation were performed where applicable. The power (1-ß) was estimated by the PASS 11 software for each individual study. RESULTS: A total of 41 eligible studies were included, concerning 11 kinds of viruses. Prevalence were significantly higher in GC for hepatitis B virus (odds ratio [OR] = 1.39, 95% confidence interval [CI] 1.11-1.75), human cytomegalovirus (OR = 2.25, 95% CI 1.14-4.43), human papillomavirus (HPV) (OR = 1.63, 95% CI 1.05-2.54), and John Cunningham virus (OR = 2.52, 95% CI 1.26-5.04). In subgroup analyses, HPV-16 infection was significantly associated with GC (OR = 2.42, 95% CI 1.00-5.83). DISCUSSION: This study demonstrated that hepatitis B virus, human cytomegalovirus, HPV, and John Cunningham virus were more prevalent in GC. However, the causal relationship between their infection and risk of GC remains inconclusive, and further investigations are required.

The Impact of Molecular Subtype on Efficacy of Chemotherapy and Checkpoint Inhibition in Advanced Gastric Cancer.

PURPOSE: We evaluated the association between molecular subtypes of advanced gastric cancer (AGC) and the efficacy of standard chemotherapy or immune checkpoint inhibitors. EXPERIMENTAL DESIGN: Patients with AGC who received systemic chemotherapy from October 2015 to July 2018 with available molecular features were analyzed. We investigated the efficacy of standard first- (fluoropyrimidine + platinum ± trastuzumab) and second-line (taxanes ± ramucirumab) chemotherapy, and subsequent anti-PD-1 therapy in patients with four molecular subtypes: MMR-D (mismatch repair deficient), EBV+, HER2+, and all negative. RESULTS: 410 patients were analyzed: MMR-D 5.9%, EBV+ 4.1%, HER2+ 13.7%, and all negative 76.3%. In 285 patients who received standard first-line chemotherapy, the median progression-free survival (PFS) times were 4.2, 6.0, 7.5, and 7.6 months and the objective response rates (ORR) were 31%, 62%, 60%, and 49% in MMR-D, EBV+, HER2+, and all-negative subtypes, respectively. Multivariate analysis showed shorter PFS in MMR-D versus all-negative patients [HR, 1.97; 95% CIs, 1.09-3.53; P = 0.022]. In second-line setting, there were no significant differences in efficacy. In 110 patients who received anti-PD-1 therapy, median PFS times were 13.0, 3.7, 1.6, and 1.9 months and the ORRs were 58%, 33%, 7%, and 13%, respectively. Twelve patients with MMR-D received subsequent anti-PD-1 therapy and showed longer PFS compared with that in 10 (83%) patients who received earlier-line chemotherapy. CONCLUSIONS: MMR-D might result in shorter PFS with first-line chemotherapy for AGC. Subsequent anti-PD-1 therapy achieved higher ORR and longer PFS than prior chemotherapy in most patients with MMR-D, supporting the earlier use of immune checkpoint inhibitors.

Clinicopathological evidence of hepatitis B virus infection in the development of gastric adenocarcinoma.

Gastric cancer (GC) is one of the infection-related cancers. Helicobacter pylori and Epstein-Barr virus (EBV) were established risk factors for GC. Recently, there are several reports showing the inconsistent association between hepatitis B virus (HBV) infection and the development of GC. To explore the relationship between HBV infection and the development of GC, we designed a meta-analysis of previous epidemiological studies, a hospital-based case-control study, followed by an immunohistochemistry (IHC) assay of HBV-exposed GC samples. We found that HBV infection was associated with an increased risk of GC based on the meta-analysis. No significant association between HBV infection and GC was detected according to our hospital-based case-control study. Histological examination showed that the gastric epithelium positive for HBx demonstrated a higher nuclear-cytoplasmic ratio compared to those HBx-negative cells. HBx and HBcAg were expressed more in tumors than those in normal counterparts in HBV-exposed subjects, and PD-L1 was lower in GC tissues from HBV carriers than those in HBV clearances. In conclusion, HBV infection may contribute to a higher risk for GC based on the meta-analysis and to the morphological atypia of gastric epithelium by the histological assessment, and GC patients among HBV carriers showed lower expression of PD-L1 may lose the chance for immune checkpoint blockade therapy.

Epstein-Barr virus is absent in gastric superficial neoplastic lesions.

Epstein-Barr virus (EBV) has been associated with about 9% of all gastric carcinomas, but its role in gastric carcinogenesis remains unclear since there is lack of evidence of EBV presence in pre-neoplastic lesions of gastric mucosa. This study intends to determine the prevalence of EBV in gastric dysplasia and superficial neoplasia to clarify whether EBV infection is an early or late event in gastric cancer development. This retrospective study included a total of 242 gastric lesions from 199 consecutive patients who were referred for endoscopic resection. The histological classification of lesions includes 137 low- and high-grade dysplasia and 105 superficial carcinomas. EBV infection was investigated by EBER-ISH. Results showed that EBV was not detected in any epithelial cells of any case with dysplasia or superficial carcinomas, although we observed the presence of a small number of EBV-infected lymphocytes in 2.1% of all lesions. These results showed that EBV is not present in gastric dysplasia neither in superficial carcinomas suggesting that EBV carcinogenesis is a late event in well/moderately differentiated gastric carcinogenesis.

Distribution of the Epstein-Barr virus in the normal stomach and gastric lesions in Thai population.

The Epstein-Barr virus (EBV) is one of the infectious agents found in stomach tissue. Recently, EBV-associated gastric carcinoma (EBVaGC) was classified as a new subtype of gastric carcinoma. To date, there is a lack of knowledge about the distribution and prevalence of EBV infection in both the normal stomach and various gastric lesions, including EBVaGC, in the Thai population. In this study, we detected EBV in the normal stomach (NS; n = 19), chronic gastritis (CG; n = 36), intestinal metaplasia (IM; n = 40), gastric dysplasia (GD; n = 15), and gastric adenocarcinoma (GC; n = 33) by polymerase chain reaction (PCR) amplification of the latent membrane protein (LMP1) gene of EBV. EBV-PCR amplification was positive in 42.1%, 36.1%, 22.5%, 13.3%, and 33.3% of NS, CG, IM, GD, and GC, respectively. For further clarification in EBVaGC, we performed EBV-encoded small RNA in situ hybridization (EBER-ISH) in PCR-positive cases of GD and GC. Four GC cases were EBER-ISH positive (12.1%), while both GD cases were EBER-ISH negative. In addition, we determined the distribution of the EBV strain (type A or B) based on EBNA3C sequence and EBV variants based on LMP1 variation (wild-type and 30-bp deletion variants; wt-LMP1 or del-LMP1). The results showed that type A and wt-LMP1 were the most prevalent in all lesions. In conclusion, EBV is common in both the NS and gastric lesions, and the frequency of EBVaGC was 12.1% in Thai patients.

Epstein-Barr virus positivity among surgically resected intramucosal gastric cancer.

Epstein-Barr virus-associated gastric cancer (EBV-GC) accounts for approximately 8% of gastric cancers. However, little is known regarding intramucosal EBV-GC. The present study aimed to evaluate endoscopic and clinicopathological characteristics of intramucosal EBV-GC. Pathological data of 172 patients with 173 intramucosal gastric cancers who received gastrectomy with lymph node dissection were obtained for review. EBV-encoded small RNA in situ hybridization (EBER-ISH) was carried out using a tissue microarray block. Eight intramucosal early gastric cancers (4.6%) were EBER-ISH positive in which no cases had any lymph node metastasis. Macroscopic types were either depressed or flat, dominant histology was mixed type of moderate and poorly differentiated adenocarcinoma. In detail, histological features of "lace pattern" or "lymphocyte infiltration into the stroma or cancer nests" were observed.

Rapamycin enhances lytic replication of Epstein-Barr virus in gastric carcinoma cells by increasing the transcriptional activities of immediate-early lytic promoters.

Epstein-Barr virus (EBV), a human herpesvirus, is linked to both epithelial and lymphoid malignancies. Induction of EBV reactivation is a potential therapeutic strategy for EBV-associated tumors. In this study, we assessed the effects of rapamycin on EBV reactivation in gastric carcinoma cells. We found that rapamycin upregulated expression of EBV lytic proteins and increased the viral proliferation triggered by the EBV lytic inducer sodium butyrate. Reverse transcription-qPCR, luciferase activity assays, chromatin immunoprecipitation and western blotting were employed to explore the mechanism by which rapamycin promotes EBV reactivation. Our results showed that rapamycin treatment resulted in increased mRNA levels of EBV immediate-early genes. Rapamycin also enhanced the transcriptional activities of the EBV immediate-early lytic promoters Zp and Rp by strengthening Sp1 binding. Repression of the cellular ataxia telangiectasia-mutated/p53 pathway by siRNA-mediated knockdown of the ataxia telangiectasia-mutated gene significantly abrogated virus reactivation by rapamycin/sodium butyrate treatment, indicating that the ataxia telangiectasia-mutated/p53 pathway is involved in rapamycin-promoted EBV reactivation. Taken together, these findings demonstrate that rapamycin might have the potential to enhance the effectiveness of oncolytic viral therapies developed for EBV-associated malignancies.

MiR-22 sustains NLRP3 expression and attenuates H. pylori-induced gastric carcinogenesis.

Chronic inflammation is the primary cause of gastric cancer (GC). NLRP3, as an important inflammasome component, has crucial roles in initiating inflammation. However, the potential roles of NLRP3 in GC is unknown. Here, we show that NLRP3 expression is markedly upregulated in GC, which promotes NLRP3 inflammasome activation and interleukin-1ß (IL-1ß) secretion in macrophages. In addition, NLRP3 binds to cyclin-D1 (CCND1) promoter and promotes its transcription in gastric epithelial cells. Consequently, NLRP3 enhances epithelial cells proliferation and GC tumorigenesis. Furthermore, we identify miR-22, which is constitutively expressed in gastric mucosa, as a suppressor of NLRP3. MiR-22 directly targets NLRP3 and attenuates its oncogenic effects in vitro and in vivo. However, Helicobacter pylori (H. pylori) infection suppresses miR-22 expression, while enhances NLRP3 expression, and that triggers uncontrolled proliferation of epithelial cells and the emergence of GC. Thus, our research describes a mechanism by which miR-22 suppresses NLRP3 and maintains homeostasis of gastric microenvironments and suggests miR-22 as a potential target for the intervention of GC.

First demonstration of equid gammaherpesviruses within the gastric mucosal epithelium of horses.

Horses commonly develop gastric mucosal ulcers, similar to humans, a condition known as equine gastric ulcer syndrome (EGUS) that can lead to poor performance and lost training time and care expenses. Unlike humans, however, an infectious bacterial cause of ulcers has not been conclusively identified. Herpesviruses, while well-established causative agents of diseases such as cold sores, genital lesions, and certain types of cancer, have also been implicated in the development of a subset of gastric ulcers in humans. The presence of equid herpesviruses in the gastrointestinal tract and their potential contribution to EGUS has not been evaluated. Here, we provide the first evidence of equid gammaherpesviruses 2 and 5 (EHV-2 and -5) within the epithelium of the gastric mucosa of horses. These viruses were initially detected by a nested PCR screen of gastric tissue samples obtained from client- and university-owned horses with and without ulcers; however, no association with EGUS was found in this limited sample set. We then validated a highly sensitive in situ hybridization (ISH) assay and used this assay to characterize the distribution of these viruses in necropsy gastric tissue samples from five racehorses. Analyses revealed frequent EHV-2 and EHV-5 co-infections within the gastric mucosal epithelium, regardless of the ulcer status. These results are the first to demonstrate the presence of equid gammaherpesviruses in the gastric mucosa of horses and warrants further investigation to determine the contribution of these viruses to the development of EGUS and/or other gastrointestinal diseases.

Cytokine expression associated with Helicobacter pylori and Epstein-Barr virus infection in gastric carcinogenesis.

Helicobacter pylori and Epstein-Barr virus (EBV) infection, and associated cytokines are involved in gastric carcinogenesis. We investigated the expression of these cytokines and their relationship with clinicopathological characteristics. The study included specimens from 207 patients with gastric adenocarcinoma, 56 with chronic gastritis, 32 with metaplasia, and 30 with low-grade epithelial dysplasia. Tissue microarrays were constructed and immunohistochemical staining for IL-1ß, IL-6, IL-10, IL-17, p16, p21, TNF-α, and TNFR1 was performed. EBV and H. pylori infection status was determined. IL-1ß, IL-6, IL-17, p16, and p21 protein expression was significantly higher in adenocarcinoma cases than in the other cases (p < 0.05). EBV was only noted in adenocarcinoma (13 cases, 6.3%). The H. pylori infection rate in adenocarcinoma was significantly higher than that in the other cases (p < 0.005). IL-6 expression was associated with improved survival (p < 0.05), whereas IL-17 expression was associated with decreased survival (p < 0.05). IL-6 expression was inversely associated with angioinvasion, and disease stage (p < 0.05), whereas IL-17 expression was associated with disease stage (p < 0.05). IL-10 expression was correlated with IL-1ß and TNF-α expression, and p16 expression was correlated with IL-17 and EBV status. Our results indicate that IL-6 and IL-17 are associated with gastric carcinogenesis and may be considered prognostic factors.